Processes of making North American ginseng fractions, products containing them, and uses as immunomodulators

ABSTRACT

The invention is directed to chemical processes of preparing fractions from North American ginseng ( Panax quinquefolium ) and pharmaceutical compositions containing these fractions. The products of the present invention may be used to stimulate the production of cytokines and/or antibodies, or as therapeutics targeted at conditions characterized by low immunity, such as the common cold, influenza, chronic fatigue syndrome, AIDS and cancer.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 10/186,733, filed Jul. 2, 2002 now abandoned, which is a divisionalof U.S. application Ser. No. 09/581,161, filed Jul. 27, 2000, now U.S.Pat. No. 6,432,454, which is the U.S. national phase of PCT/US98/25724,filed Dec. 11, 1998, which claims the benefit of provisional applicationNo. 60/069,534 filed Dec. 12, 1997. All of these applications areincorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to chemical processes of making fractions fromNorth American ginseng (Panax quinquefolium) and compositions containingthese fractions. The products of the present invention may be used tostimulate the production of antibodies, or as therapeutics targeted atconditions characterized by low immunity, such as the common cold,influenza, chronic fatigue syndrome, AIDS, cancer, etc. The products ofthe present invention may also be used as a supplement for cancerpatients undergoing chemotherapy or radiation therapy, which is known tocause serious suppression of the immune system.

BACKGROUND OF THE INVENTION

For hundreds of years, the use of certain non-toxic agents such asherbal compounds has been widely accepted for a variety of physiologicalconditions, especially in the Orient. Panax ginseng C. A. Meyer is thebest known traditional Chinese medicine. The important pharmacologicalactivities of ginseng extracts, alone or in combination with otherdrugs, include alleviation of renal impairment, inhibition ofcarcinogenesis and prevention of stress. There are also a number ofreports on the influence of ginseng on the immunological responsivenessof the individual. Some immunomodulatory properties that have beenreported include enhancement of host resistance against infection,anti-inflammatory effect, inhibition of tumor growth, as well asmodulation of some basic immune function at the cellular level.

American ginseng, Panax quinquefolium, is another specie of ginsengwhich has gained popularity as a health supplement having manybeneficial health effects. Several groups of scientists have attemptedto isolate and elucidate the structure of the polysaccharides present inginseng. Some of the polysaccharides have been demonstrated to be activein modulating the immune system.

A series of studies on the isolation, characterization, and biologicalevaluation of ginseng polysaccharides was carried out by Tomoda's groupin Kyoritsu College of Pharmacy, Japan. In one set of studies, ginsengpolysaccharides were fractionated based on their acidity. Two acidicpolysaccharides having immunological activities have been isolated fromroot of Korean ginseng (Panax ginseng)^([1,2]). The sliced roots wereextracted with hot water. The extract was treated withcetyltrimethylammonium bromide (CTAB) in the presence of sodium sulfate.The precipitate was separated, dialyzed, and applied to a Sephadex G-25column, DEAE-Sephacel (Pharmacia) column to give two purepolysaccharides, which were designated as ginsenan PA and ginsenan PB.Gel chromatography on Toyopearl HW-55F gave the values of 1.6×10⁵and5.5×10⁴ for the molecular weight of ginsenan PA and ginsenan PB,respectively. Quantitative analyses showed that ginsenan PA contained21.3% arabinose, 53.4% galactose, 2.0% rhamnose, 16.0% galacturonic acidand 2.7% glucuronic acid. The molar ratio of these component sugars was11:22:1:6:1. Ginsenan PB contained 11.0% arabinose, 32.2% galactose,8.1% rhamnose, 39.9% galacturonic acid, and 5.0% glucuronic acid. Themolar ratio was 3:7:2:8:1. Both polysaccharides showed markedreticuloendothelial system-potentiating activity in a carbon clearancetest, and pronounced anti-complementary activity and alkalinephosphatase-inducing activity in a dose dependent manner.

In another study^([3]), an additional two polysaccharides were isolatedfrom the supernatant of the above extract treated with CTAB, i.e., thesupernatant was poured into ethanol. The precipitate was separated andapplied to columns of DEAE-Sephadex A-25 and Sephadex G-25 to giveanother two pure polysaccharides, designated S-IA and S-IIA. Gelchromatography on Toyopearl HW-55F gave the values of 5.6×10⁴ and1.0×10⁵ for the molecular weight of S-IA and S-IIA respectively.Ginsenan S-IA contains 42.3% arabinose, 50.8% galactose and 6.9%galacturonic acid with the molar ratio of 8:8:1. Ginsenan S-IIA iscomposed of 42.0% L-arabinose, 32.6% galactose, 6.2% glucose, and 19.2%galacturonic acid. The molar ratio is 15:10:2:5.

Several polysaccharide fractions from leaves and roots of Panax ginsenghave been separated by Yamada's group in the Oriental Medicine ResearchCenter of the Kitasato Institute, Japan. The chemical properties andbiological activities were investigated and compared.^([4])

Ginseng roots and leaves from China, after treated with ethanol toremove their ginsenosides, were extracted with water, and the residuewere extracted with 0.5 M NaOH to give water-soluble and alkalinesoluble polysaccharide fractions (designated as GR-2 and GL-2 for thewater-soluble fractions and GRA-2 and GLA-2 for the alkaline-solublefractions, respectively). Based on the acidity of the componentpolysaccharides, all fractions were further fractionated into stronglyacidic (designated as GR-3, GL-3, GRA-3, and GLA-3), weakly acidic(designated as GR4, GL4, GRA4, and GLA4), and neutral polysaccharidefractions (designated as GR-5, GL-5, GRA-5, and GLA-5), by treatmentwith cetyltrimethylammonium. The roots contained larger amount ofpolysaccharide than the leaves. The strongly acidic polysaccharidefractions from the roots had a high content of uronic acid, even higherthan 50% . Similar component sugars were detected from all fractions.They were rhamnose, arabinose, galactose, glucose, galacturonic acid,and glucuronic acid. Galacturonic acid was the main uronic acidcomponent.

The fraction with highest anti-complementary activity, GL-3, was furtherfractionated with columns of DEAE-Sephadex, Sepharose CL-6B, DEAEToyopearl, and ethanol precipitation to give fractions designated asGL-PI through GL-PIV.^([5]) All fractions contained 32–44% uronic acid.Fraction PI had the highest molecular weight of 50,000. PI and PIIconsisted mainly of Rha, Gal, and GalA, and PIII contained Fuc inaddition, whereas PIV consisted of Gal, Glc, and GalA. A detailedstructural determination was performed.

An anti-ulcer pectic polysaccharide (GL-BIII) was isolated from weaklyacidic polysaccharide fraction GL-4 by chromatography on DEAE SepharoseCL-6B and Sepharose CL-6B. It was mainly composed of Rha, Ara, Man, Gal,Glc, GalA, and GlcA in the molar ratio of 3:4:2:10:1:7:4. Detailedstructural determination was performed.^([6])

Another macrophage Fc receptor expression-enhancing polysaccharide(GL4IIb2) was separated from GL-4 by anion-exchange chromatography onDEAE-Sepharose CL-6B. Chemical analysis showed that the sample contained65% total carbohydrate and 33.7% uronic acid. The composition analysisand structural determination were performed.^([7])

Another Panax ginseng extract with anticomplementary activity, G-115,was studied by the same group.^([8]) G-115 was fractionated in order tocharacterize the active substances for anticomplementary and mitogenicactivities. The most potent anticomplementary activity was observed inthe crude polysaccharide fraction, G-115G, whereas the water-solubledialyzable fraction, G-115E, showed the most potent mitogenic activity.G-115G was further purified by precipitation with cetyltrimethylammoniumbromide, anion-exchange chromatography on DEAE-Sepharose and gelfiltration on Sepharose CL4B, and a major potent anticomplementarypolysaccharide, G-115l1-IIa-2-3 was obtained. This polysaccharide washomogeneous. Its molecular weight was estimated to be 3.68×10⁵. Itconsisted mainly of arabinose, galactose and glucose in addition tosmall amounts of galacturonic acid, glucuronic acid and rhamnose.

Ginseng polysaccharides were isolated from Korean, Chinese, and Japaneseginseng by Hikino's group at the Pharmaceutical Institute, TohokuUniversity, Japan. The hypoglycemic activities of the ginsengpolysaccharides have been tested. The composition and some structurefeature have been elucidated.^([9-14])

Three polysaccharides, quinquefolans A through C, were isolated fromAmerican ginseng.^([14]) Their molecular weights were estimated to behigher than 2.0×10⁶ by gel chromatography over Sephacryl S-500. Theneutral sugar components were mannose and glucose (molar ratio, 1.0:2.3)for quinquefolan A, mannose and glucose (1.0:5.5) for quinquefolan B,and xylose for quinquefolan C. The acidic sugar components inquinquefolans A through C were found to be 10.8, 11.7, and 7.1%respectively. The content of peptide moieties in these glycans was 2.7,2.9, and 2.3% for quinquefolans A through C respectively. All of thesepolysaccharides showed hypoglycemic effects in normal andalloxan-reduced hypoglycemic mice.

An acidic polysaccharide with the molecular weight of 150,000, calledginsan, was isolated from Panax ginseng by a research group at theLaboratory of Immunology, Korean Cancer Center Hospital, Seoul,Korea.^([15]) This polysaccharide was composed of 3.7% protein and 47.1%hexose (glucose and galactose) and 43.1% uronic acid (galacturonicacid). Ginsan induced the proliferation of T cells and B cells andgenerated lymphokine activated killer cells from both natural killer andT cells through endogenously produced multiple cytokines.^([16])

Miao et al. from Northeast Normal University of China has isolatedpolysaccharides from American ginseng. The purification and structuralanalysis were performed.^([17])

The biological activities of polysaccharides from American ginseng havebeen investigated by a research group in Norman Bethune University ofMedical Science.^([18, 19]) They found that polysaccharides fromAmerican ginseng (PPQ) enhanced lymphocyte transformation. The effect ofpolysaccharide from Panax quinquefolium (PPQ-1) on cytokine productionfrom murine spleen lymphocyte in vitro was studied. The data suggestthat PPQ-1 regulates immune function.

SUMMARY OF THE INVENTION

The present inventors have found that certain American ginseng extractshave immunoregulating properties. CVT-E002, and purified fractions PQ₂and PQ₂₂₃ therefrom, specifically stimulates murine spleen cells toproliferate B cells, which subsequently produce a large amount ofantibody. The fractions also increase serum immunoglobulin (e.g., totalIgG) levels and stimulate macrophages to produce IL-1, IL-6 and TNF-α.These fractions may be used for the prevention or treatment of generalinfection and other immune deficiency associated diseases.

Therefore, the present invention is directed to processes of preparingginseng fractions PQ₂, PQ₂₂₃ and CVT-E002 from samples of Americanginseng.

Specifically, a process of preparing ginseng fraction PQ₂ comprises:

-   -   combining American ginseng with a first solvent comprising an        alcohol and heating the resulting solution at a temperature of        about 80–100° C. for a time period of about 2–4 hours to produce        a first ginseng solution;    -   thereafter separating the first ginseng solution to produce an        alcohol/ginseng solution and a first ginseng residue;    -   thereafter combining the first ginseng residue with water and        heating the resulting solution at a temperature of about        80–100° C. for a time period of about 2–4 hours to produce a        ginseng residue solution;    -   thereafter separating the ginseng residue solution to produce a        second ginseng residue and a first aqueous extract solution        containing a first ginseng extract;    -   providing a second aqueous extract solution which comprises at        least a part of the first ginseng extract, wherein in the second        aqueous extract solution the proportion of the first ginseng        extract to water is about 1:18 to 1:22;    -   thereafter combining the second aqueous extract solution with a        second solvent comprising an alcohol, wherein the proportion of        the second solvent to water is about 1:1 to 3:5, to produce a        first precipitate and a first supernatant;    -   thereafter combining the first supernatant produced in the        previous step with a third solvent comprising an alcohol,        wherein the proportion of the third solvent to first supernatant        is about 3:2 to 3:1, to produce a second precipitate and a        second supernatant; and    -   isolating the second precipitate to produce ginseng fraction        PQ₂.

A process of preparing ginseng fraction PQ₂₂₃ comprises:

-   -   providing ginseng fraction PQ₂, as described above;    -   fractionating the ginseng fraction PQ₂ to produce a first        elution fraction and a second elution fraction, wherein the        first elution fraction corresponds to a carbohydrate peak        observed between 35 and 50 ml of elution volume and the second        elution fraction corresponds to a carbohydrate peak observed        between 50 and 85 ml of elution volume, as determined by gel        filtration chromatography using the following materials:        -   (1) a chromatographic column containing a matrix of a            spherical cross-linked co-polymer of allyl dextran and            N,N′-methylenebisacrylamide, having a bed dimension of            16×600 mm, a bed volume of 120 ml, and a fractionation range            (MW) of 5000 to 250,000 for globular proteins and 1000 to            80,000 for dextrans, and        -   (2) an elution buffer of Tris-HCl containing 0.1 N HCl and            0.3 M NaCl at a pH of 7.0; and    -   isolating and combining the first elution fraction and the        second elution fraction to produce ginseng fraction PQ₂₂₃.

A process of preparing ginseng fraction CVT-E002 comprises:

-   -   combining American ginseng with a first solvent comprising an        alcohol in a proportion of about 7–9 ml of first solvent per        gram of ginseng and heating the resulting solution at a        temperature of about 80–100° C. for a time period of about 2–4        hours, to produce a first ginseng solution;    -   thereafter separating the first ginseng solution to produce an        alcohol/ginseng solution and a first ginseng residue;    -   thereafter combining the first ginseng residue with water in a        proportion of about 7–9 ml of water per gram of ginseng residue        and heating the ginseng residue solution at a temperature of        about 80–100° C. for a time period of about 2–4 hours, to        produce a ginseng residue solution;    -   thereafter separating the ginseng residue solution to produce a        second ginseng residue and an aqueous extract solution        containing a ginseng extract; and    -   drying the aqueous extract solution to produce ginseng fraction        CVT-E002.

The present invention also includes ginseng fractions PQ₂, PQ₂₂₃ andCVT-E002, which are prepared according to the processes described above.

The invention further includes ginseng fractions having specificcarbohydrate contents.

A first ginseng fraction has a carbohydrate content which comprisesabout 2–6 mol % rhamnose, about 41–49 mol % galacturonic acid, about12–18 mol % glucose, about 16–22 mol % galactose and about 12–19 mol %arabinose.

A second ginseng fraction has a carbohydrate content which comprisesabout 3–8 mol % rhamnose, about 36–44 mol % galacturonic acid, about 2–7mol % glucose, about 25–33 mol % galactose and about 17–25 mol %arabinose.

A third ginseng fraction has a carbohydrate content which comprisesabout 0.5–5 mol % rhamnose, about 11–22 mol % galacturonic acid, about40–60 mol % glucose, about 10–19 mol % galactose and about 11–19 mol %arabinose.

The invention also includes pharmaceutical compositions, comprising theginseng fractions of the invention, in combination with apharmaceutically acceptable carrier.

The invention further includes the use of a ginseng fraction accordingto the invention, alone or in combination with another medicament, inthe preparation of a pharmaceutical composition suitable for treating acondition characterized by low immunity.

The invention also includes the use of a ginseng fraction of theinvention to stimulate the production of IL-1, IL-6 and/or TNF-α incells.

The invention further includes the use of a ginseng fraction accordingto the invention to stimulate the in vitro or in vivo production ofimmunoglobulins.

Also included is the use of a ginseng fraction according to theinvention to activate B-lymphocyte proliferation and antibody productiontherefrom.

The invention also includes a method of treating a conditioncharacterized by low immunity in a patient in need thereof, comprisingadministering to the patient a condition treating effective amount of aginseng fraction according to the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a plot of elution volume against log molecular weight forstandard dextran samples eluted on a chromatographic column.

FIG. 2 shows a chromatogram of ginseng fraction CVT-E002.

FIG. 3 shows a chromatogram of ginseng fraction PQ₁.

FIG. 4 shows a chromatogram of ginseng fraction PQ₂.

FIG. 5 shows a chromatogram of ginseng fraction PQ₃.

FIG. 6 shows a chromatogram of ginseng fraction PQ₂₂₃.

FIG. 7 shows the effect of ginseng fraction PQ₂₂₃ on mouse splenocyte Bcell proliferation.

FIG. 8 shows the effect of ginseng fraction PQ₂₂₃ on mouse splenocyte Tcell proliferation.

FIG. 9 shows the specificity of the effect of ginseng fraction PQ₂₂₃ onmouse splenocyte T and B cell proliferation.

FIG. 10 shows the effect of ginseng fraction PQ₂₂₃ on in vitro antibodyproduction by mouse spleen.

FIG. 11 shows the enhancement of plaque forming cells (PFC) in miceinjected with ginseng fraction PQ₂₂₃.

DETAILED DESCRIPTION OF THE INVENTION

A process of preparing ginseng fraction PQ₂ comprises:

(a) combining American ginseng with a first solvent comprising analcohol and heating the resulting solution at a temperature of about80–100° C. for a time period of about 2–4 hours to produce a firstginseng solution;

(b) thereafter separating the first ginseng solution to produce analcohol/ginseng solution and a first ginseng residue;

(c) thereafter combining the first ginseng residue with water andheating the resulting solution at a temperature of about 80–100° C. fora time period of about 2–4 hours to produce a ginseng residue solution;

(d) thereafter separating the ginseng residue solution to produce asecond ginseng residue and a first aqueous extract solution containing afirst ginseng extract;

(e) providing a second aqueous extract solution which comprises at leasta part of the first ginseng extract, wherein in the second aqueousextract solution the proportion of the first ginseng extract to water isabout 1:18 to 1:22;

(f) thereafter combining the second aqueous extract solution with asecond solvent comprising an alcohol, wherein the proportion of thesecond solvent to water is about 1:1 to 3:5, to produce a firstprecipitate and a first supernatant;

(g) thereafter combining the first supernatant produced in step (f) witha third solvent comprising an alcohol, wherein the proportion of thethird solvent to first supernatant is about 3:2 to 3:1, to produce asecond precipitate and a second supernatant; and

(h) isolating the second precipitate to produce ginseng fraction PQ₂.

The alcohol in each of the first solvent, second solvent and thirdsolvent comprises an alcohol which is inert to the ginseng and is easilyseparated from the desired product. Those of skill in the art wouldreadily be able to select alcohols which meet these requirements.Preferably the alcohol in each case independently comprises a saturatedor unsaturated C₁–C₆ alcohol. More preferably, the alcohol in each caseindependently comprises ethanol or methanol.

In each of steps (a) and (c), it is preferred that the resultingsolution is heated for a time period of about 3 hours. It is alsopreferred that in step (a) the first solvent and the ginseng arecombined in a proportion of about 7–9 ml of first solvent per gram ofginseng, most preferred about 8 ml of first solvent per gram of ginseng.In step (c), it is preferred that the water and the first ginsengresidue are combined in a proportion of about 7–9 ml of water per gramof ginseng residue, most preferred about 8 ml of water per gram ofginseng residue.

Directly following step (d) in the process recited above, the firstginseng extract may optionally be concentrated. This may be accomplishedin any way according to procedures well known to those of skill in theart. For example, the first aqueous solution containing the firstginseng extract may be centrifuged (e.g., at a speed of 2500 to 10,000rpm for about 5–15 minutes). The first aqueous solution may also befiltered. Alternatively or in addition to concentrating the firstginseng extract, the first ginseng extract may be freeze dried for lateruse.

In step (e), the second aqueous extract solution comprises at least apart of the first ginseng extract. It is important that the ratio offirst ginseng extract to water in the second aqueous extract solution beabout 1:18 to 1:22, more preferably about 1:20. This may be achieved inany of a number of ways. If the first ginseng extract is concentratedand/or freeze dried following step (d), as described above, water shouldbe added to the first ginseng extract to achieve the desired ratio.Alternatively, a part of the first aqueous extract solution, or theentire first aqueous extract solution, may be used in the second aqueousextract solution. If needed, additional water may be added to achievethe desired ratio. The use of at least a part of the first aqueousextract solution which contains the desired amount of first ginsengextract would avoid the need to conduct additional concentrating orfreeze drying steps between steps (d) and (e).

In step (f), it is preferred that the proportion of the second solventto water is about 3:4.

In step (g), it is preferred that the proportion of the third solvent tofirst supernatant is about 2:1.

A process of preparing ginseng fraction PQ₂₂₃ comprises:

(a) providing ginseng fraction PQ₂, produced according to the processdescribed above;

(b) fractionating the ginseng fraction PQ₂ to produce a first elutionfraction and a second elution fraction, wherein the first elutionfraction corresponds to a carbohydrate peak observed between 35 and 50ml of elution volume and the second elution fraction corresponds to acarbohydrate peak observed between 50 and 85 ml of elution volume, asdetermined by gel filtration chromatography using the followingmaterials:

-   -   (1) a chromatographic column containing a matrix of a spherical        cross-linked co-polymer of allyl dextran and        N,N′-methylenebisacrylamide, having a bed dimension of 16×600        mm, a bed volume of 120 ml, and a fractionation range (MW) of        5000 to 250,000 for globular proteins and 1000 to 80,000 for        dextrans, and    -   (2) an elution buffer of Tris-HCl containing 0.1 N HCl and 0.3 M        NaCl at a pH of 7.0; and

(c) isolating and combining the first elution fraction and the secondelution fraction to produce ginseng fraction PQ₂₂₃.

The first elution fraction is also known as PQ₂A, and the second elutionfraction is also known as PQ₂B. These elution fractions may be isoatedseparately. Additionally, the same process as noted above may be used toproduce additional elution fractions PQ₂C (which corresponds to acarbohydrate peak observed between 95 and 110 ml of elution volume, andPQ₂D (which corresponds to a carbohydrate peak observed between 120 and250 ml of elution volume). Each of these fractions may also beseparately isolated. In addition, compositions besides PQ₂₂₃ whichcomprise two or more of fractions PQ₂A through PQ₂D may be also made.

It is preferred that ginseng fraction PQ₂ is fractionated using gelfiltration chromatography. However, any other types of fractionationknown to the skilled artisan are suitable.

The procedure for performing gel filtration chromatography is well knownby those of ordinary skill in the art, following manufacturer'srecommendations as to flow rate, sample volume and temperature at whichthe procedure should be performed. Variance of these factors within themanufacturer's specifications does not significantly affect the resultsof the chromatographic run.

Determination of carbohydrate content may be made by any procedure knownin the art. It is preferred that a microtiter plate assay be conductedfor the determination of the carbohydrate composition of the fraction.Such an assay is well known to those of skill in the art. See, forexample, Dubois et al. Anal. Chem. 28: 350–56 (1956), herebyincorporated by reference. Absorbance of the sample provided accordingto the microtiter plate assay is preferably conducted at 492 nm.

A process of preparing ginseng fraction CVT-E002 comprises:

(a) combining American ginseng with a first solvent comprising analcohol in a proportion of about 7–9 ml of first solvent per gram ofginseng and heating the resulting solution at a temperature of about80–100° C. for a time period of about 2–4 hours, to produce a firstginseng solution;

(b) thereafter separating the first ginseng solution to produce analcohol/ginseng solution and a first ginseng residue;

(c) thereafter combining the first ginseng residue with water in aproportion of about 7–9 ml of water per gram of ginseng residue andheating the ginseng residue solution at a temperature of about 80–100°C. for a time period of about 2–4 hours, to produce a ginseng residuesolution;

(d) thereafter separating the ginseng residue solution to produce asecond ginseng residue and an aqueous extract solution containing aginseng extract; and

(e) drying or concentrating the aqueous extract solution to produceginseng fraction CVT-E002.

The alcohol in the first solvent comprises an alcohol which is inert tothe ginseng and is easily separated from the desired product. Those ofskill in the art would readily be able to select alcohols which meetthese requirements. Preferably the alcohol comprises a saturated orunsaturated C₁–C₆ alcohol. More preferably, the alcohol comprisesethanol or methanol.

It is preferred that in step (a) the first solvent and the sample arecombined in a proportion of about 8 ml of first solvent per gram ofsample. It is also preferred that in step (c) the water and the firstginseng residue are combined in a proportion of about 8 ml of water pergram of ginseng residue.

In steps (a) and (c), it is preferred that the first ginseng solution isheated for a time period of about 3 hours.

The invention also includes several ginseng fractions.

A first ginseng fraction has a carbohydrate content which comprisesabout 2–6 mol % rhamnose, about 41–49 mol % galacturonic acid, about12–18 mol % glucose, about 16–22 mol % galactose and about 12–19 mol %arabinose. Preferably, the carbohydrate content comprises about 3–5 mol% rhamnose, about 43–47 mol % galacturonic acid, about 14–16 mol %glucose, about 18–20 mol % galactose and about 14–17 mol % arabinose.Most preferably, the carbohydrate content comprises about 4 mol %rhamnose, about 45 mol % galacturonic acid, about 15 mol % glucose,about 19 mol % galactose and about 15 mol % arabinose.

A second ginseng fraction in accordance with the invention has acarbohydrate content which comprises about 3–8 mol % rhamnose, about36–44 mol % galacturonic acid, about 2–7 mol % glucose, about 25–33 mol% galactose and about 17–25 mol % arabinose. Preferably, thecarbohydrate content comprises about 4–7 mol % rhamnose, about 37–42 mol% galacturonic acid, about 3–6 mol % glucose, about 27–32 mol %galactose and about 19–24 mol % arabinose. Most preferably, thecarbohydrate content comprises about 5 mol % rhamnose, about 39 mol %galacturonic acid, about 4 mol % glucose, about 29 mol % galactose andabout 21 mol % arabinose.

A third ginseng fraction according to the invention has a carbohydratecontent which comprises about 0.5–5 mol % rhamnose, about 11–22 mol %galacturonic acid, about 40–60 mol % glucose, about 10–19 mol %galactose and about 11–19 mol % arabinose. Preferably, the carbohydratecontent comprises about 1–3 mol % rhamnose, about 13–20 mol %galacturonic acid, about 42–57 mol % glucose, about 12–17 mol %galactose and about 13–17 mol % arabinose.

The invention also includes pharmaceutical compositions, comprising anyof the ginseng fractions according to the invention in combination witha pharmaceutically acceptable carrier. Those of skill in the art arefamiliar with any pharmaceutically acceptable carrier which would beuseful in this regard, and therefore the procedure for makingpharmaceutical compositions in accordance with the invention will not bediscussed in detail. Suitably, the pharmaceutical compositions may be inthe form of tablets, capsules, liquids, lozenges, lotions orsuppositories.

The invention includes the use of a ginseng fraction according to theinvention in the preparation of a pharmaceutical composition suitablefor treating a condition characterized by low immunity, such as commoncold, influenza, chronic fatigue syndrome, AIDS and cancer. The ginsengfraction may be used alone or in combination with another medicament.The ginseng fractions of the invention are especially suitable forco-administration with a chemotherapeutic agent or as a supplement toradiation therapy, since cancer patients are known to have serioussuppression of the immune system.

The invention also includes a method of treating a conditioncharacterized by low immunity in a patient in need thereof, comprisingadministering to the patient a condition treating effective amount of aginseng fraction according to the invention. Preferably, the conditionis selected from the group consisting of common cold, influenza, chronicfatigue syndrome, AIDS and cancer. Dosages of ginseng fractions inaccordance with the invention depend upon the particular condition to betreated, as well as the age, sex and general health condition of thepatient. However, suitable dosages may be found in the range between 1and 5000 mg/kg body weight per day, in between 1 and 10 daily doses. Theginseng fractions may be administered orally, via injection or infusion,topically, nasally, ocularly, vaginally or rectally.

The invention will now be further elucidated by the following Examples.

EXAMPLE 1

A First Process for Preparing Fraction CVT-E002 and Purifying thisFraction

American ginseng root was chemically extracted and purified sequentiallyto give fractions CVT-E001 and CVT-E002. An amount of CVT-E002 wasfurther purified to give fractions G₁, G₂ and G₃. The detaileddescription of the procedure is as follows.

500 grams of dried ground root of ginseng was extracted with 4 liters of85% ethanol or 3 liters of 90% methanol on a water bath at 80–85° C.while stirring for 3 hours and filtered to give an alcohol solution andresidue. The alcohol solution was concentrated and spray dried to give aproduct of total saponins (CVT-E001). The residue was extracted with 4liters of water on a water bath at 95–100° C. while stirring for 3hours. The extract was filtered through a muslin bag and centrifuged togive an aqueous solution containing CVT-E002, and the remaining residuewas discarded.

A portion of the aqueous solution was used for further purification, andan equal volume of 95% ethanol was added to the aqueous solution, whichcaused precipitation. The precipitate was centrifuged and lyophilized togive the fraction G₁. The supernatant was reduced in volume byevaporation. An equal volume of 95% ethanol was added to give the nextprecipitate, G₂, and the remaining supernatant was removed with ethanoland lyophilized to produce a powder, G₃.

The further purification of G₂ was conducted as follows. Two grams of G₂were dissolved in 80 ml of water and dialyzed against 3 volumes of waterin a Sigma D-7884. dialysis tubing with a 1200 molecular weight cut-off.The dialysis was done at 4° C. for 72 hours, collecting dialysate twiceevery 24 hours. The resulting dialysate was concentrated to 15 ml, thenprecipitated with an equal volume of methanol. The precipitate wasdissolved in water and lyophilized to yield a powder, G₂₂.

The further purification of G₂₂ was conducted as follows. 1.5 grams ofG₂₂ were dissolved in 60 ml water and then dialyzed against 600 mls ofwater in a Fisher Spectral/Por molecularporous membrane tubing with a1000 molecular weight cut-off at 4° C. The first dialysate collectedafter 24 hours was concentrated to 15 mls using an evaporator and thenlyophilized. The resulting dried powder was referred to as G₂₂₁. In asimilar manner, a second batch of dialysate was collected after 48 hoursand referred to as G₂₂₂. The retentate was concentrated and lyophilizedto give a powder called G₂₂₃.

EXAMPLE 2

A Second Process for Preparing Fraction CVT-E002 and Purifying thisFraction

An alternative process for preparing ginseng fraction CVT-E002 accordingto the invention is as follows.

1000 grams of dried ground root of American ginseng was extracted with 8liters of 85% ethanol on a water bath at 95–100° C. while stirring for 3hours and filtered to give an alcohol solution and residue. The residuewas combined with water (1:8) on a hot water bath with continuingagitation for 3 hours. After cooling to room temperature, the mixturewas filtered. The filtrate was centrifuged at 5000 rpm for 10 min. Thesupernatant was concentrated and freeze dried to give extract CVT-E002.The amount of CVT-E002 produced by this method is approximately the sameas produced according to Example 1, i.e., about 10% of the weight of theoriginal raw ginseng.

A portion of CVT-E002 was further fractionated as follows. 1600 ml of95% ethanol was added to a solution of 100 grams of CVT-E002 powder in2000 ml of water. The precipitate was isolated as PQ₁. The supernatantwas concentrated to 500 ml. Another portion of 95% ethanol (1000 ml) wasadded to this concentrated solution again to give second precipitatefraction. The precipitate was isolated and freeze dried to give fractionPQ₂. The supernatant was concentrated and freeze dried to give PQ₃.

EXAMPLE 3

Mitogenic Activity Test

Different fractions of American ginseng at various levels ofpurification were selected by screening on the basis of their mitogenicactivity of lymphocytes in vitro. Though mitogenicity is considered arather artificial event in relation to the normal events occurring inthe immune system in vivo, mitogens provide good indications of possibleeffector function.

The test method for mitogenic activity is described as follows: Balb/Cor C57B1/6J mice were used for the test. Balb/C mice were obtained fromthe Health Sciences Laboratory Animal Services facility (University ofAlberta, Edmonton, Canada). C57B1/6J mice were obtained from the JacksonLaboratory (Bar Harbor, Me.). Mice from 7 to 10 weeks old were age andsex matched for each experiment. Mice were killed by cervicaldislocation. Spleens were removed by using aseptic techniques and werecrushed between the frosted ends of two glass slides. After washing bycentrifugation in Hanks Balanced Salt Solution (HBSS), the cells weresuspended in RPMI 1640 medium pH 7.4 (Gibco, Grand Island, N.Y.)containing 10% fetal bovine serum (Flow Labs), 50 mM mercaptoethanol(ICN Pharmaceuticals, Plainview, N.Y.), and penicillin-streptomycin(Gibco). Cultures were set up in triplicate in 96-well flat-bottomedLinbro plates at 1.25×10⁶ cells per ml final concentration. Experimentalgroups were set up with testing fractions previously filter-sterilizedand dissolved in HBSS. Control cultures consisted of a group withoutmitogen and groups with 20 μg/ml phytohemagglutinin (PHA) or 25 μg/mllipopolysaccharide (LPS). It is known that PHA specifically stimulatesthe T-type of spleen cells while LPS stimulates the B-type of spleencells. The cultures were incubated at 37° C. in a humidified 5% CO₂atmosphere for 72 hours. Four hours before the end of the 72 hourincubation, 1 μCi of tritiated thymidine (New England Nuclear, Boston,Mass.) was added to each well. The cells were harvested with anautomated sample harvester (Skatron, Va.) and then incorporatedradioactivity was assayed by scintillation counting. The results werecalculated as a % of control (mean±standard deviation, in triplicate).

The different extraction conditions shown in Examples 1 and 2, such asconcentration of CVT-E002 in water and the volume of ethanol used forprecipitation, affect the further fractionation of the CVT-E002. Theproducts obtained from these two processes were compared for mitogenicactivity. The results are shown in the following Tables. Each test wasperformed in triplicate with three batches.

TABLE 1 Mitogenic activity of G₁, G₂, and G₃ obtained from procedureaccording to Example 1 Activity (% control) Fractions Batch 1 Batch 2Batch 3 G₁ 4090 ± 1644 2845 ± 1318 4446 ± 977 G₂ 3015 ± 1284 4101 ± 16713146 ± 318 G₃ 1791 ± 573  1735 ± 96  365 ± 26

TABLE 2 Mitogenic activity of PQ₁, PQ₂, and PQ₃ obtained from procedureaccording to Example 2 Activity (% control) Fractions Batch 1 Batch 2Batch 3 PQ₁ 2448 ± 454 3169 ± 431 2492 ± 138 PQ₂  6427 ± 1609  6467 ±1593  7034 ± 1834 PQ₃ 1313 ± 288 1522 ± 342 1622 ± 222

As Tables 1 and 2 indicate, the change in extraction procedure betweenExamples 1 and 2 correlates with a transfer of biological activity fromthe G₁ fraction to the PQ₂ fraction.

EXAMPLE 4

Fractionation of PQ₂ and a Process for Preparing Fraction PQ₂₂₃

CVT-E002, PQ₁, PQ₂ and PQ₃ according to Example 2 were furtherfractionated according to their molecular weight distribution by gelfiltration chromatography over a HiPrep 16/60 Sephacryl S-200 highresolution column (Pharmacia Biotech, Cat. No. 17-1166-01), whichcontains a matrix of a spherical cross-linked co-polymer of allyldextran and N,N′-methylenebisacrylamide, has a bed dimension of 16×600mm, a bed volume of 120 ml, and a fractionation range (MW) of 5000 to250,000 for globular proteins and 1000 to 80,000 for dextrans. Dextransamples (MW=71.4 k, 37.5 k, 19.5 k and 9.5 k) were purchased from Sigmaand were used as standard samples. 5 mg of each sample was dissolved in1 ml of water, loaded onto the column, and eluted with Tris-HCl buffercontaining 0.1 N HCl and 0.3 M NaCl, pH 7.0, flow rate 0.3 ml/min andenvironmental temperature 4° C. A volume of eluate was collected as anumber of individual 5 ml portions.

Total carbohydrate content of each individual portion of eluate wastested with a microtiter plate assay for total carbohydrate content.This is a modification of a method described in Dubois et al. Anal.Chem. 28: 350–56 (1956). The total carbohydrates in the eluate arederivatized in order to be detectable at a certain absorbance spectrum.D-glucose was used as the standard sample, and other materials used wereconcentrated sulfuric acid (98%) and 5% phenol. The polystyrenemicrotiter plates were purchased from SARSTDT (Quebec, Canada). To eachwell of a microtiter plate, 40 μl of a standard sample solutioncontaining 1 to 10 μg of D-glucose was applied and 40 μl of 5% phenolwas added and mixed. 200 μl of concentrated sulfuric acid was thencarefully added. After mixing reagent and individual portion of eluatewith a multichannel pipette, the plate was incubated under 80° C. for 1hour. After cooling to room temperature, the absorbance of the samplewas measured at 492 nm on a Multiskan microtiter plate reader. The datawas saved and the gel filtration chromatography results were plottedusing the Microsoft Excel program.

A standard curve was created by running a series of standard dextrans(Sigma) with known molecular weights over the same column (FIG. 1). Thechromatograms for CVT-E002 and lots 21 of PQ₁, PQ₂ and PQ₃ are shown inFIGS. 2–5. In each Figure, points on the x-axis show the elution volumein terms of one-fifth of the actual volume. In order to get a true valueof elution volume, therefore, the results in the Figures must bemultiplied by five.

The gel filtration chromatogram of CVT-E002 (FIG. 2) showed mainly threepeaks. The molecular weight for first peak (elution fraction 7–10) wasestimated to be 70,000 or higher according to the standard curve (FIG.1). The molecular weight for the third peak (elution fraction 18–22) wasestimated to be about 1,000. A small broad second peak (elution fraction10–17) was also observed. The ratio of these three peaks was57.2:8.5:34.4.

The chromatogram of PQ₁ (FIG. 3) showed one main peak (elution fraction7–10), which corresponds to the high molecular weight fraction ofCVT-E002, and a minor peak (elution fraction 18–21). The ratio of thetwo peaks was 86.8:13.2.

The chromatogram of PQ₂ (FIG. 4) showed three main peaks. The peakdesignated A (elution fraction 7–10) and the peak designated C (elutionfraction 19–22) correspond to the two main peaks of CVT-E002. Anotherbroad peak, designated B (elution fraction 10–17), was observed betweenpeaks A and C. The molecular weight for this peak was estimated to beabout 2,000–60,000. The ratio of the three peaks A–C was 34.1:45.7:20.3.A region of small peaks, designated D (elution fraction 24–50, a portionof which is shown in FIG. 4), was also observed.

The chromatograph of PQ₃ (FIG. 5) showed only one main peak (elutionfraction 18–21), which corresponded to the low molecular weight fractionof CVT-E002.

Fractions A, B, C and D of PQ₂ were collected and freeze dried. Thedried powders were renamed as PQ₂A, PQ₂B, PQ₂C and PQ₂D, respectively.Fractions A and B were also combined, and the combined fraction wascalled PQ₂₂₃ (a chromatograph of which is shown in FIG. 6). Themitogenic activity of PQ₂A, PQ₂B, PQ₂C and PQ₂D were tested and comparedwith the activity of fractions PQ₂₂₃, PQ₂ and CVT-E002. The results areshown in the following Table.

TABLE 3 Mitogenic activity of American ginseng fractions (³H-thymidineincorporation as % of control) Sample Dose (μg/ml) Activity (% ofControl) Control n/a 100 PHA  20 1334.5 ± 158*   LPS  25  3227 ± 177**PQ₂A 100  3002 ± 147** PQ₂B 100  947 ± 42** PQ₂C 100 186.5 ± 10.5* PQ₂D100 201 ± 5** CVT-E002 100 1395.5 ± 159.5* PQ₂ 100 2321.5 ± 8.5**  PQ₂₂₃100 3237 ± 13** * and ** represent P < 0.05 and P < 0.01, respectively.n = 3.

As the results indicate, the order of the potency is PQ₂₂₃>PQ₂>CVT-E002.

EXAMPLE 5

Chemical Determination of Total Carbohydrate and Protein Composition ofCVT-E002, PQ₁, PQ₂, PQ₃, and PQ₂₂₃

Three batches of each fraction CVT-E002, PQ₁, PQ₂, PQ₃, and PQ₂₂₃ wereobtained, and the total carbohydrate and protein composition weremeasured in each fraction. The total carbohydrate content was determinedby the phenol-sulfuric method, as known in the art. The protein contentwas assayed by Lowry method, as known in the art, and bovine serumalbumin was used as standard. The results are shown in the followingTable.

TABLE 4 Comparison of Protein and Carbohydrate content of fractionsProtein Total Sample Content (%) Carbohydrate (%) E002-1 9.32 66.4E002-2 7.38 69.9 E002-5 8.31 60 PQ₁-24 2.6 90.3 PQ₁-25 2.6 93.6 PQ₁-272.9 96.1 PQ₂-24 5.6 52.6 PQ₂-25 5.4 46.8 PQ₂-27 5.3 43.6 PQ₃-24 7.9 52.6PQ₃-25 8.5 64.2 PQ₃-27 7.1 68.0 PQ₂₂₃-7 5.8 78.0 PQ₂₂₃-8 2.8 81.0PQ₂₂₃-9 2.9 85.2

EXAMPLE 6

Chemical Determination of Monosaccharide Composition of CVT-E002, PQ₁,PQ₂ and PQ₂₂₃

Since total carbohydrate content mainly represented the amount ofpolysaccharides and oligosaccharides, the molar ratios ofmonosaccharides for certain fractions were also determined.

Lots of fractions CVT-E002, PQ₁, PQ₂ and PQ₂₂₃ were subjected to acidcatalyzed hydrolysis to liberate their structural units,monosaccharides. A qualitative and quantitative analysis of theliberated monosaccharides was performed by HPLC after they werederivatized with MPP (3-methyl-1-phenyl-2-pyrazolin-5-one). The detailedmethods are described as follows: a sample solution in 2 N HCl washeated at 95–100° C. for 6 h. The mixture was neutralized with NaOHsolution. An internal standard was added. MeOH and aqueous NaOH solution(0.3 N) were then added to this mixture. This was then stirred at 50–60°C. for 8 hr (or at room temperature for one week). The mixture wasdiluted (1:10) with water and analyzed by HPLC. The MPP-derivatizedsamples were analyzed with a C-18 column, with an eluent of 18%acetonitrile in 0.1 M phosphate buffer (pH 7), at a flow rate of 1ml/min, UV detector at 245 nm. The following Table shows the molar ratioof monosaccharides of CVT-E002, PQ₁, PQ₂ and PQ₂₂₃. Trace amounts ofglucuronic acid was found in some CVT-E002, PQ₁ and PQ₂ samples.

TABLE 5 Monosaccharide Composition of CVT-E002, PQ₁, PQ₂ and PQ₂₂₃ (% inmolar) Galacturonic Sample Rhamnose Acid Glucose Galactose ArabinoseE002-1 2.8 17.9 53 13.3 13 E002-2 1 13.1 56.7 12.7 15.2 E002-3 2.9 20.442.1 16.5 16.3 PQ₁-21 0.67 8.27 82.6 4.57 3.72 PQ₁-22 0.48 6.38 85 4.563.55 PQ₁-23 0.48 8.24 80.7 4.68 4.72 PQ₂-22 3.6 45.1 15.4 19.8 16.2PQ₂-26 3.8 44.2 15.6 19 14.4 PQ₂-27 3.8 44.2 15.6 19 14.4 PQ₂-28 4 45.313.8 20.5 16.3 PQ₂₂₃-7 5.5 40.8 4.1 28.2 20.3 PQ₂₂₃-8 5.4 38.1 3.9 29.522.1 PQ₂₂₃-9 5.4 36.7 5.4 28.8 22.7

As the result indicate, the order of glucose content is as follows:PQ1>CVT-E002>PQ2>PQ3; and the content of the galacturonic acid is asfollows: PQ2>PQ223>CVT-E002>PQ1.

EXAMPLE 7

Immune Regulation of Ginseng Fractions

1. Macrophage Activity

The effects of CVT-E002, PQ₂, G₂ and PQ₂₂₃ on murine macrophages werestudied in vitro using peritoneal exudate macrophages from C57B1/6 micewhich preferentially mount a cell-mediated response and Balb/c micewhich preferentially mount an antibody-mediated response. IL-1, IL-6 andTNF-α production were measured after macrophages were stimulated with100 μg/ml of test samples. The methods are described as follows.

A. Preparation of Macrophages from Peritoneal Exudate Cells and CellSupernatant.

-   1). 1 ml of 3% thioglycollate was injected into the murine    peritoneal cavity and peritoneal exudate macrophages were harvested    from the peritoneal cavity after 3 days.-   2). Cell suspension was washed twice with Hanks buffer by    centrifugation at 1100 rpm at 4° C. for 5 min.-   3). Macrophages were suspended in RPMI-10% FBS medium.-   4). Cells were counted and diluted to a final concentration of    10⁶/ml with RPMI-10% FBC medium.-   5). Macrophages were cultured in 10×10⁶/10 ml RPMI-10% FBS at 37° C.    for 2 hrs. Supernatant was discarded and the precipitated    macrophages were washed with 10 ml of PBS twice.-   6). Macrophages were harvested and re-suspended in RPMI-10% FBS.-   7). Macrophage (5×10⁵) were cultured with 10 mg/ml of LPS or 100    mg/ml of test sample for 24 or 48 hrs, after which time the    supernatants were collected and filter sterilized for the bioassay    (IL-1, IL-6 and TNF-α). Experiments were carried out in triplicate.    B. IL-1 Determination in Supernatant.

The NOB1 cell line is a TK-variant of the EL4 cell line that generatesCTLL growth stimulating activity (IL-2) in response to IL-1. Since NOB1cells do not incorporate ³HTdR, a response to IL-1 can be measured interms of uptake by the CTLL line. The procedure is described as follows.

-   1). 10⁴ CTLL cells were combined with 4×10⁴ NOB1 cells in 200 μl of    5% FBS in IMDM in each well of a 96-well flat-bottom microtiter    plate. Serially diluted IL-1 test samples of 4, 8, 16 or 32-fold or    serial dilutions of IL-1 standards were applied. The final volume in    each well was 200 μl. Each sample was prepared in triplicate.-   2). Plates were incubated for 24 hr in a 37° C. 5% CO₂ humidified    incubator.-   3). [³H]-thymidine was added for the last 5 hr of incubation.-   4). Cells were harvested and [³H]-thymidine incorporation was    measured by liquid scintillation counting.-   5). IL-1 concentration was calculated. The results are shown in the    following Table.

TABLE 6 Effect of ginseng fractions on IL-1 production by murinemacrophages Macrophages¹ Production of stimulated with . . . IL-1²(pg/ml) cells only 15.7 ± 1.0  LPS  27.7 ± 1.9** CVT-E002 18.1 ± 1.2*PQ₂ 19.5 ± 0.9* G₂ 17.7 ± 0.7* PQ₂₂₃ 19.9 ± 1.4* ¹Peritoneal exudatemacrophages 5 × 10⁵/ml were cultured with 100 μg/ml samples, 10 μg/mlLPS for 48 hrs and culture supernatant was harvested. ²Assay of IL-1 wasperformed using NOB1 and CTLL cell lines and results were expressed asmean ± SD *P < 0.05; **P < 0.01

The results show that CVT-E002, PQ₂, G₂ and PQ₂₂₃ significantlystimulated macrophages from C57B1/6 mice to produce IL-1. All testsamples showed a similar potency of stimulating IL-1 production.

C. Determination of IL-6

The proliferation of a murine B cell hybridoma cell line, B9, isIL-6-dependent. B9 cells were cultured in a series of microwellscontaining decreasing concentration of the test samples. The procedurewas as follows.

-   1). The number of B9 cells in an aliquot removed from the stock    culture flask was counted at their log-phase of growth.-   2). The cells were centrifuged for 5 min in a tabletop centrifuge at    180×g, 4° C., and the pellet was resuspended in 10 ml of complete    RPMI-10 medium. The procedure was repeated twice and the cells were    resuspended in complete RPMI-10 medium at 2×10³ cells/ml.-   3). 100 μl of washed cells (2×10³ cells/ml) was added to each well    of a 96-well microtiter plate.-   4). 100 μl of 2-fold serial dilutions of the test samples were added    to each well, reserving two or three rows of the plate for IL-6    standards.-   5). The plates were incubated for 72 hr in a humidified 37° C., 5%    CO₂ incubator.-   6). [³H]-thymidine was added and plates were incubated for 4 hr at    37° C.-   7). The cells were harvested and [³H]-thymidine incorporation was    determined using a liquid scintillation counter.-   8). IL-6 concentration was calculated. The results are as shown in    the following Table.

TABLE 7 Effect of ginseng on IL-6 production by murine macrophagesMacrophages¹ Production of IL-6² (pg/ml) stimulated with . . . C57B1/6mice Balb/c mice cells only 71.7 ± 7   102 ± 1.8  LPS 14312.5 ± 269.4** 22496.8 ± 2381.2** CVT-E002 181.5 ± 2.1** 185.5 ± 10*   PQ₂  219.3 ±30.3** 243.4 ± 6.4** PQ₂₂₃  2058.3 ± 137.9**  2387.2 ± 301.6**¹Peritoneal exudate macrophages 5 × 10⁵/ml were cultured with 100 μg/mlsamples, 10 μg/ml LPS for 24 hrs and culture supernatant was harvested.²Assay of IL-6 was performed using B9 cell line and results wereexpressed as mean ± SD *P < 0.05; **P < 0.01

CVT-E002, PQ₂ and PQ₂₂₃ increased the production of IL-6 in supernatantof macrophages from C57B1/6 and Balb/c mice. The potency isPQ₂₂₃>PQ₂>CVT-E002.

D. Measurement of TNF-α

The following protocol employed TNF-sensitive, actinomycin D-treatedmurine L929 fibroblasts to quantify TNF activity in supernatants derivedfrom cell cultures.

-   1). 4×10⁴ L929 cells in 50 μl of IMDM-5% FSC were added into each    well.-   2). 50 μl of test sample was added to the second well of each row    (column 2). Two-fold serial dilutions were made by gently mixing the    contents of well 2 and transferring 50 μl from well 2 into well 3.    The contents of well 3 were mixed gently, and 50 μl from well 3 was    transferred into well 4. This procedure was continued through    well 12. Finally, the contents of all wells in column 12 were gently    mixed and 50 μl was discarded from each well. At this point, all    wells contained 50 μl.-   3). 50 μl of actinomycin D solution was added to each well (2 μl    ml).-   4). The plates were incubated for 24 hr at 37° C. in 5% CO₂ in air.-   5). 5 μl of Neutral Red was added to each well and the plates were    incubated for 2.5 hours.-   6). All supernatants in each well were quickly and carefully rinsed    and emptied, and plates were washed with 200 μl of PBS twice.-   7). The PBS was aspirated and 100 μl of 50% ethanol in 0.05M NaH₂PO₄    was added to each well and shaken for 5 min at room temperature.-   8). Each well was read immediately with a microtiter plate reader at    an absorbance of 570 nm.-   9). TNF-α concentration was calculated. The results are shown in the    following Table.

TABLE 8 Effect of ginseng on TNF-α production by murine macrophagesMacrophages¹ Production of TNF-α² (pg/ml) stimulated with . . . C57B1/6mice Balb/c mice cells only 6.1 ± 1.9 4.8 ± 0.7  LPS  55.2 ± 1.6**  71.4± 15.5** CVT-E002  19.7 ± 2.5** 17.9 ± 2.8** PQ₂ 3.5 ± 0.8 3.9 ± 0.2  G₂6.1 ± 1.2 7.2 ± 0.1* PQ₂₂₃  16.9 ± 2.5** 15.2 ± 2.2** ¹Peritonealexudate macrophages 5 × 10⁵/ml were cultured with 100 μg/ml samples, 10μg/ml LPS for 48 hrs and culture supernatant was harvested. ²Assay ofTNF-α was performed using L929-8 cell line and results were expressed asmean ± SD of TNF-α production by macrophages. *P < 0.05; **P < 0.01

TNF-α production was induced by CVT-E002 and PQ₂₂₃ in supernatant ofmacrophages from Balb/c or C57B1/6 cell lines. TNF-α in the supernatantof macrophages from Balb/c mice was significantly stimulated by G₂. PQ₂seemed not to stimulate TNF-α production by macrophages.

2. Serum Immunoglobulin Production

Serum immunoglobulin production (total IgG) increased 21% in mice fedwith CVT-E002, 26% in mice fed with PQ₂ and 31% in mice fed with PQ₂₂₃compared with mice fed with water. The potency is PQ223>PQ2>CVT-E002.Results are shown in Table 9.

TABLE 10 In vivo immunoglobulin production by mice (n = 15) Mice fedwith . . . IgG (μpg/ml) ± SD Water 275.16 ± 32.34  CVT-E002 333.01 ±50.88** PQ₂ 346.81 ± 31.39** PQ₂₂₃ 400.46 ± 54.48**3. Specificity of Mitogenic Activity of PQ₂₂₃

Well known mitogens such as PHA or LPS show specificity as to the typeof cells they stimulate. It is important to know which specific type ofspleen cells are stimulated by ginseng fractions. We therefore separatedT cells by an affinity column and enriched for B cells by T celldepletion.

A. T-Cell Enrichment

A single cell suspension of spleen cells was layered on lymphocyte-M(Cedarlane Labs) to remove red blood cells. The lymphocytes were thenpassed through an affinity chromatography column (Biotex Co. Ltd.,Edmonton, Alberta) which removes B cells by adherence to the mouseimmunoglobulin coated beads. The eluant consisted of an enrichedpopulation of T cells. Determination of the percentage of T cellsrelative to other cell types was done by the viability test usinganti-thy 1.2 monoclonal antibody and complement treatment.

B. B-Cell Enrichment

After removal of red blood cells from the spleen cell population, thelymphocytes were incubated with monoclonal anti-thy 1.2 antibody for onehour at 37° C. Low-tox rabbit complement was then added and incubatedfor half an hour at 4° C. This procedure depleted the cell suspension ofT cells, resulting in an enriched B cell population.

C. Culturing with PQ₂₂₃

B or T cells were cultured with varying doses of PQ₂₂₃, and theresponses were measured in terms of tritiated thymidine incorporation.FIGS. 7 and 8 show the proliferation effect of PQ₂₂₃ on relativelypurified B and T cells, respectively. Control groups included cellstreated with LPS, a B cell-specific mitogen, and PHA, a T cell-specificmitogen. PQ₂₂₃ was found to be B cell-specific (see FIG. 9).

4. The Effect of PQ₂₂₃ on Antibody Production In Vitro

Spleen cells were cultured for 72 hours in the presence of varying dosesof PQ₂₂₃. Control cultures consisted of a group to which 25 μg of LPSwas added, and another to which no mitogen was added. The supernatantswere tested for the presence of soluble immunoglobulins using the EnzymeLinked Immunosorbent Assay (ELISA). The supernatant containing solubleimmunoglobulins was serially diluted in 0.1M tris buffer (pH 9).Microtiter plates were coated with this supernatant and incubated at 4°C. overnight. The plates were washed with PBS Tween three times, then100 μl of the antibody-enzyme conjugate Goat F(ab′)2 anti-mouse Ighorseradish peroxidase (Tago Inc., Burlingame Calif.) was added at a1:1000 dilution. The plate was incubated for one hour at roomtemperature. The plate was again washed three times with PBS, then 100μl of ABTS peroxidase substrate (Kirkegaard and Perry Lab Inc.) wasadded to each well. One hour later absorbance was measured at awavelength of 405 nm by a Flow Multiscan ELISA reader.

FIG. 10 shows that 50 μg/ml PQ₂₂₃ stimulated the production ofimmunoglobulins comparable to that of 25 μg LPS. A higher concentrationof PQ₂₂₃, 500 μg/ml, did not elicit a higher degree of antibodyproduction. This dose response curve is consistent with theproliferative responses of heterogeneous spleen cell population andpurified B cells. These results were based on four experiments.

5. In Vivo Antibody Plaque Forming Cells

Three concentrations of PQ₂₂₃ were i.v injected to three groups of 3mice each. A control group was injected with a balanced salt solution.At the end of seven days of intravenous treatments with PQ₂₂₃, the micewere immunized with 0.2 ml of a 10% SRBC suspension in HBSS. Five daysafter immunization the mice were sacrificed and single cell suspensionsfrom the spleens were prepared and adjusted to 4×10⁶ cells per ml. A 0.1ml aliquot of this cell suspension was mixed with 0.3 ml of a 10% SRBCsolution guinea pig complement and 0.5 ml RPMI medium. Seventymicroliters of this mixture was transferred to a Cunningham chamber. Thechamber was sealed with paraplast wax and was incubated at 37° C. forone hour prior to counting of plaque forming cells. All PFC levels areexpressed as per one million spleen cells. FIG. 11 shows that all threedoses significantly enhanced antibody production in mice injected withthe compound.

In summary, the results of the present studies indicate that the ginsengfractions of the invention activate macrophages to produce cytokinessuch as IL-1, IL-6 and TNF-α. They also activate lymphocytes,particurlarly B-lymphocyte proliferation and antibody production. Theoverall humoral mediated immune system was stimulated indicating thepreventive effects on infections. The reported action of TNF-αproduction includes antiviral and anti-tumor benefits. TNF-α has alsobeen reported to be of therapeutic benefit in the treatment of a varietyof parasitic infections.

The following citations referred to in the Background of the Inventionare hereby incorportated by reference.

-   1. Tomoda et al. Biol. Pharm. Bull., 16, 22–5 (1993).-   2. Tomoda et al. Biol. Pharm. Bull., 17, 1287–91 (1994).-   3. Tomoda et al. Biol. Pharm. Bull., 16, 1087–90 (1993).-   4. Gao et al. Planta Medica, 55, 9–12 (1989).-   5. Gao et al. Carbohydr. Res., 181, 175–87 (1988).-   6. Kiyohara et al. Carbohydr. Res., 263, 89–101 (1994).-   7. Shin et al. Carbohydr. Res., 300, 239–49 (1997).-   8. Yamada et al. Phytotherapy Res, 9, 264–9 (1995).-   9. Konno et al. Planta Medica, 50, 434–6 (1984).-   10. Hikino et al., unpublished.-   11. Oshima et al. J. Ethnopharmacology, 14, 255–9 (1985).-   12. Konno et al. Int. J. Crude Drug Res., 25, 53–6 (1987).-   13. Konno et al. J. Ethnopharmacology, 14, 69–74 (1985).-   14. Oshima et al. J. Natural Products, 50, 188–90 (1987).-   15. Lee et al. Anticancer Res., 17, 323–32 (1997).-   16. Kim et al. Planta Medica, 64, 110–5 (1998).-   17. Miao et al. Shengwu Huaxue ZaZhi, 9, 6104 (1993).-   18. Ma et al. Baiqiuen Yike Daxue Xuebao, 23, 236–8 (1997).-   19. Zhu et al. Zhongguo Yaolixue Tongbao, 13, 76–8 (1997).

1. A pharmaceutical compositions, wherein said pharmaceuticalcomposition comprises ginseng fraction CVT-E002 and a pharmaceuticallyacceptable carrier.
 2. A method of preparing a pharmaceuticalcomposition suitable for treating a condition characterized by lowimmunity, wherein said method comprises combining an effective amount ofginseng fraction CVT-E002 and, optionally, an effective amount ofanother medicament that is suitable for treating a conditioncharacterized by low immunity with at least one pharmaceuticallyacceptable excipient to produce a pharmaceutical composition suitablefor treating a condition characterized by low immunity.
 3. The method ofclaim 2, wherein the condition is selected from the group consisting ofcommon cold, influenza, chronic fatigue syndrome, AIDS and cancer.
 4. Amethod of stimulating the production of IL-1, IL-6 and/or TNF-α incells, wherein said method comprises contacting said cells with aneffective amount of ginseng fraction CVT-E002.
 5. A method of activatingB-lymphocyte proliferation and antibody production resulting from saidB-lymphocyte proliferation in a patient in need of such proliferation,wherein said method comprises administering to the patient an effectiveamount of ginseng fraction CVT-E002.
 6. A method of treating a conditioncharacterized by low immunity in a patient in need thereof, comprisingadministering to the patient an effective amount of ginseng fractionCVT-E002.
 7. The method of claim 6, wherein the condition is selectedfrom the group consisting of common cold, influenza, chronic fatiguesyndrome, AIDS and cancer.
 8. A method of stimulating the in vitroproduction of immunoglobulins in cells, wherein said method comprisescontacting cells with an effective amount of ginseng fraction CVT-E002.9. A method of stimulating the in vivo production of immunoglobulins ina subject in need thereof, wherein said method comprises administeringto the subject an effective amount of ginseng fraction CVT-E002.